Antiplasmodial Activity and Cytotoxicity of Plants Used in Traditional Medicine of Iran for the Treatment of Fever

Malaria is the most serious parasitic disease and one of the oldest recorded diseases in the world. Because of the resistance of malaria parasites to current drugs, it is necessary to discover new antiplasmodial drugs. Traditional medicine is one of the important sources of new antiplasmodial drugs. In this study, twenty methanolic extracts from different parts of sixteen medicinal plants used in traditional medicine of Iran for the treatment of “Nobeh fever” and/ or fever were screened for in-vivo antiplasmodial activity against Plasmodium berghei and cytotoxic effect on Madin–Darby bovine kidney cells (MDBK). Eleven species (55%) were found to have antiplasmodial activity. Methanolic extract from Rosa damascena Mill. reduced parasitemia by 57.7% compared to untreated control mice at intra-peritoneal (i.p.) injection doses of 10 mg/Kg per day for 4 days. This is the first report that mentioned in-vivo antiplasmodial activity of Rosa damascena Mill.


Introduction
Malaria is the most serious parasitic disease and one of the oldest recorded diseases in the world. The name «mal» «aria»(meaning «bad air» in Italian) comes from 18 century Italian word (1). It affects 219 million people per year worldwide and is the cause of 660,000 deaths per year (2).
In recent years, the increasing resistance of malaria parasites to drugs or malaria vectors to pesticides, led to discover and develop new antiplasmodial and new pesticides agents (1, 3).
Traditional medicines have been used to treat malaria for thousands of years and are the source of the two main groups (artemisinin and quinine derivatives) of modern antiplasmodial drugs (3). The vast majority of people on this planet still rely on their traditional materia medica for their everyday health care needs (4). Besides, herbal medicines are widely believed to be safe and efficacious. Therefore, the potential of plants traditionally used to inhibit parasite growth without host toxicity must be assured (5). One of the methods which are applied for the safety evaluation is in-vitro cytotoxicity assay against normal cells (6).
Tertian, quartana and tropical are three different forms of malaria according to the type

Plants selection
Fever attacks are the most common symptoms of malaria. So, the term "fever" was searched in the ancient medicinal books of Iran for selecting the plants.

Preparation of plant samples
Seven plant species out of sixteen plants used for treating nobeh fever and other fevers were collected from different places of Iran. The others were purchased from herbal market (Attari) ( Table 1). The plants were identified by the taxonomist and voucher specimens were deposited at Traditional Medicine and Materia Medica Research Center (TMRC) herbarium.

Plant extraction
The selected parts of the collected plants were air-dried in shadow. Plants were crushed into powder using a hammer mill and extracted by maceration of 10 g of powdered dried material of parasite and period of fever (7). The term of malaria did not exist in the ancient medicinal books but ancient physicians knew this disease. Avicenna, the Iranian philosopher and physician, (980-1037AD) about 1000 yr ago described the clinical features of an intermittent febrile attack with 4-12 h period of cold, hot, and sweating stages which is actually the characters of paroxysm of malaria (8). Among the symptoms of malaria, fever attacks are the most common symptom. Physicians of traditional medicine were divided fever into three categories: "Yomiyyeh fever (ephemeral fever)" "Degh fever (hectic fever)" "Ofouni fever (infectious fever)". Infectious fever occurs when Akhlat (structural components) receive external heat. Ofouni fever is periodical like malaria. Physician of traditional medicine said "Nobeh fever" to this fever (9).
In the present study, the in-vivo antiplasmodial and in-vitro cytotoxic effect of 20 extracts from 16 medicinal plants that were used in traditional medicine of Iran against "Nobeh fever" and other fevers (10-17) were evaluated.

Biological assays In-vivo antiplasmodial assay
To carry out the screening of the extracts, the Peters' 4-day suppressive test against Plasmodium berghei infection in mice was employed (18-21). All the procedure was accepted by Shahid Beheshti University Ethics Committee and in accordance with the principles for laboratory animal use and care in the European Community guidelines. On day 1 (D 0 ), all experimental adult male albino mice weighing 20-25 g were infected by intra-peritoneal (i.p.) injection with 1×10 infected erythrocytes. The mice were randomly divided into groups of five per cage and treated during consecutive days with 10 mg/ Kg of the sample by i.p injection for 4 days (on days D 0 , D 1 , D 2 and D 3 ). Two control groups were used in this experiment, one treated with chloroquine as a positive control while the other group was kept untreated as a negative control. On day 5 (D 4 ) of the test, thin blood smears were prepared and blood films were fixed with methanol. The blood films were stained with Giemsa, and then microscopically examined. Percentage of parasitaemia was counted based on infected erythrocytes calculated per 1000 erythrocytes.

In-vitro cytotoxic assay
Methanolic extracts of all plants were screened for cytotoxic with the Madin-Darby bovine kidney normal cells (MDBK). Suspension containing 1×10 cell/mL was seeded into 96well micro plates. After 24 h, cells were washed and maintained with different concentrations of extract for 3 days, at 37 °C, under 5% CO 2 atmosphere. The initial concentration of extracts was 100 µg/mL in DMSO, which was serially diluted in complete culture medium with two fold dilutions to give six concentrations (100 -3.125 µg/mL). The cytotoxicity of the plant extracts was determined using the colorimetric methylthiazole tetrazolium (MTT) assay (19-23) and scored as a percentage of absorbance reduction at 570 nm of treated cultures versus untreated control cultures. IC 50 values on cell growth were obtained from the drug concentration-response curves. 5-Fluorouracil (5-Fu) was examined as a positive control.

Results and Discussion
The results of the cytotoxicity and the in-vivo antiplasmodial of plant extracts were reported in Table 2. Most of them exhibited no significant cytotoxicity. The Convolvulus scammonia L. extract was found to be cytotoxic. Twenty extracts were prepared from the selected parts of the sixteen plants species. Eleven extracts (55%) showed in-vivo antiplasmodial activity. Rosa damascena Mill. showed significant suppression of parasitemia (57.7%). Three plants, Carthamus tinctorius L., Cotoneaster nummularia Fisch. & Mey. and Cassia angustifolia Vahl. showed moderate antiplasmodial activity. Rosa damascena Mill. commonly known as rose having several pharmacological properties including anti-HIV, antibacterial, antioxidant, antitussive, hypnotic, anti-diabetic and relaxant effect on tracheal chains have been reported for this plant. Several components were isolated from flowers, petals and hips (seed-pot) of R. damascena including terpenes, glycosides, flavonoids, and anthocyanins. This plant contains carboxylic acid, myrcene, vitamin C, kaempferol and quarcetin. Flowers also contain a bitter principle, tanning matter, fatty oil and organic acids. The essential oil of R. damascena, contains eighteen compounds represented more than 95% of the total oil. The identified compounds were; β-citronellol (14.5-47.5%), nonadecane (10.5-40.5%), geraniol (5.5-18%), andnerol and kaempferol were the major components of the oil. Analyses of rose absolute showed that phenyl ethylalcohol(78.38%), citrenellol (9.91%), nonadecane (4.35%) and geraniol (24)(25)(26). In traditional medicine of Iran, R. damascena Mill. was used to treat depression, headache, strengthening the heart, skin problems, wounds and quartan fever (16).
The main goal of this work was to investigate the potential antiplasmodial properties of some plants used in traditional medicine of Iran against nobeh fever and/ or fever. Among sixteen plant species, only three of them (Althaea officinalis Ms. Leila Farivar for her assistance to carry out this work.
We would like to express our special gratitude for late Farzaneh Naghibi. She is remembered for her tremendous intellect, integrity, energy and enthusiasm devoted to promotion of scientific research particulary in this work.

References
L., Cichorium intybus L. and Cordia myxa L.) have been previously investigated for their antimalarial activity (27)(28)(29)(30). So, this is the first report of the antiplasmodial properties of the plants. The next step will be to phytochemical investigation and survey on the mode of action.